T cells were isolated from PBMCs with EasySep™ Human T cell Isolation Kit per manufacturer’s instructions (StemCell Technologies). Post-isolation purity was routinely 96-98%. Isolated T cells (0.5-1 x 105 per well) or unseparated PBMCs (1 x 105 per well) were stimulated for 2-3 days either with Dynabeads™ Human T Activator CD3/CD28 antibody coated beads (Thermo Fisher) at 4:1 bead:cells ratios or with plate-bound anti-CD3 and soluble anti-CD28 at the indicated concentrations in the presence of varying concentrations of SMAC mimetics or DMSO control. Cells were incubated at 37°C and 6.0% CO2. In some experiments, cells were labeled with CFSE or Cell Trace e450 (CTe450) proliferation dye (Thermofisher) prior to culture. Cells were incubated with 10 uM CFSE or CTe450 in 1x PBS/0.1%BSA for 15 minutes at 37°C, then washed three times before culture.
Dynabeads human t activator cd3 cd28 antibody coated beads
Manufactured by Thermo Fisher Scientific
Dynabeads™ Human T Activator CD3/CD28 antibody coated beads are a type of magnetic bead coated with antibodies specific to the CD3 and CD28 receptors on human T cells. These beads can be used to activate and expand human T cells in vitro.
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2 protocols using dynabeads human t activator cd3 cd28 antibody coated beads
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T cell activation and proliferation assay
2
T cell Activation and Proliferation
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T cells were isolated from PBMCs with EasySep™ Human T cell Isolation Kit per manufacturer's instructions (StemCell Technologies). Isolated T cells (0.5-1 x 10 5 per well) or unseparated PBMCs (1 x 10 5 per well) were stimulated for 48-72 hours either with Dynabeads™ Human T Activator CD3/CD28 antibody coated beads (Thermo Fisher) at 2:1 or 4:1 bead:cells ratios or with plate-bound anti-CD3 and soluble anti-CD28, each at 2 ug/ml in the presence of varying concentrations of IAP antagonists. Cells were incubated at 37°C and 6.0% CO2. In some experiments, cells were labeled with CFSE or Cell Trace e450 proliferation dye (Thermofisher) prior to culture. Cells were incubated with 10uM CFSE or CTe450 in 1x PBS/0.1%BSA for 15 minutes at 37°C, then washed three times before culture.
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